Anti-pathogenic composition useful in blood preservation

ABSTRACT

The present invention includes a method for reducing viral, bacterial, protozoan, fungal and other parasitic contamination from a biological solution. Biological solutions include, but are not limited to, solutions comprising blood, a blood component, cell culture or a component of a cell culture. In accordance with the present invention, there is provided, a method of inactivating a pathogen in blood or blood product in a container, comprising contacting at least one of said blood, blood product and container with a composition of the present invention. In accordance with the present invention, there is provided, a medical device comprising at least a surface treated with an anti-pathogenic composition of the present invention or containing at least an anti-pathogenic composition of present invention. In accordance with the present invention, there is provided, an anti-pathogenic composition for use in disinfecting fluids and biological tissues and surfaces contaminated with fluids and/or biological tissues, which comprises an anti-pathogenic amount of at least one quaternary ammonium compound in association with an acceptable carrier. A preferred anti-pathogenic composition of the present invention further comprises a bisguanidine compound. In accordance with the present invention, there is provided a method for inhibiting in vitro or ex vivo infection or replication of human immunodeficiency virus in a biological fluid, comprising treating said biological fluid with an effective inhibiting amount of a bis-guanidine compound or a derivative thereof, and at least one quaternary ammonium compound in combination with a pharmaceutically carrier, such as DMSO.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the prior benefit of U.S. provisionalapplication Ser. No. 60/331,806, filed Nov. 21, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pathogenic compositions for use inblood and blood products, and blood containers, such as a blood donationbag. The present invention also relates generally to a method ofprocessing and disinfecting human blood products. More particularly,this invention relates to a method of disinfecting whole blood, bloodcells, plasma proteins, and plasma so that they may be used safely andeffectively for diagnostic, therapeutic or research purposes. Thepresent invention also relates to applications vis the blood circulatorysystem for controlling the pathological state produced by a pathogen,typically a viral, bacterial, protozoan, fungal or parasitic agent (atany stage of the life-cycle of the parasitic agent).

2. Description of Prior Art

The development of plastic blood collection bags has facilitated theseparation of donated whole blood into its various components andanalogous products, thereby making these different blood products (e.g.,platelet concentrates) available as a transfusion product. With thepassage of time and accumulation of research and clinical data,transfusion practices have changed greatly. One aspect of currentpractice is that whole blood is rarely administered; rather, patientsneeding red blood cells are given packed red cells, patients needingplatelets are given platelet concentrate, and patients needing plasmaare given plasma.

However, the increase in transfusing blood products finds a concomitantincrease in the transmission of disease to the transfusion recipient.There is an existing and great need to assure a disease-free bloodsupply.

Blood products from human and animal donors are widely used fortherapeutic, diagnostic and experimental purposes. A persistent problemassociated with using blood products from human and animal donors isthat these products are subject to contamination by blood-borne virusesand other micro-organisms such as bacteria. Of particular threat areviruses that appear to cause various forms of hepatitis, including thehepatitis B virus, the non-A, non-B hepatitis virus or viruses. Othersof interest are cytomegalovirus and Epstein-Barr virus.

Viruses linked with the incurable and often fatal disease known asacquired immune deficiency syndrome (AIDS) are caused by a retrovirus orgroup of retroviruses (HIV, HIV-1, and HIV-2). The most common cause ofAIDS is thought to be HIV-1.

The threat of hepatitis, AIDS, and bacterial transmission throughtransfusion and administration of blood products is not limited to bloodcells but extends to the administration of plasma and plasma fractionssuch as Factor VIII concentrates, Factor IX concentrates, gammaglobulin, and anti-thrombin III.

Disinfecting whole blood and blood products, including red blood cells,plasma, and plasma fractions with disinfectants strong enough tosignificantly inactivate viruses, bacteria and other organisms hasgenerally been discounted because active agents strong enough toinactivate the pathogen typically damage cellular blood constituents orinactivate plasma and plasma protein factions. Additionally, thepresence of any residual disinfectant in the blood product to betransfused could be hazardous to the recipient of the transfusion.

A typical component separation procedure used in the United States, thecitrate-phosphate-dextrose-adenine (CPDA-1) system, utilizes a series ofsteps to separate donated blood into three components, each componenthaving substantial therapeutic and monetary value. The proceduretypically utilizes a blood collection bag which is integrally attachedvia flexible tubing to at least one, and preferably two or more,satellite bags. Using centrifugation, whole blood may be separated bydifferential sedimentation into such valuable blood components asplasma, packed red cells (PRC), platelets suspended in clear plasma(platelet-rich plasma, or PRP), platelet concentrate (PC), andcryoprecipitate (which may require extra processing).

Commonly used systems other than CPDA-1 include Adsol, Nutricell, andSAG-M. In these latter systems, the collection bag contains onlyanti-coagulant, and the nutrient solution may be pre-placed in asatellite bag. This nutrient solution is transferred into the PRC afterthe PRP has been separated from the PRC, thereby achieving a higheryield of plasma and longer storage life for the PRC. Improvements incurrent practices of viral marker screening and donor self-exclusion arecontinuously increasing the safety of the blood supply. However, despitethese practices, a risk of transmission of pathogens with thetransfusion of cellular components of blood remains since currentscreening tests do not screen for rarely occurring or as yet unknowntransfusion transmissible pathogens (Dodd, R. Y. New Engl. J. Med.327:419-421 (1992); Soland, E. M., et al. J. Am. Med. Assoc.274:1368-1373 (1995); Schreiber, G. B., et al. New Engl. J. Med.334:1685-1690 (1996)).

To combat the deficiencies associated with screening practices, the useof sterilization procedures of blood, red blood cell concentrates(RBCC), and other blood-derived components hold promise for eliminatingpathogen transmission. In this connection, various approaches have beenused to sterilize blood cells, the most efficacious so far usephotochemical methods (Ben-Hur, E. and B. Horowitz Photochem. Photobiol.62:383-388 (1995); Ben-Hur, E. and B. Horowitz AIDS 10:1183-1190(1996)). The most promising photochemical methods employ the use ofphthalocyanines (which are activated by light in the far red (660-700nm)) for sterilization of RBCC (Horowitz, B., et al. Transfusion31:102-108 (1991); Ben-Hur, E., et al. J. Photochein. Photobiol. B:Biol.13:145-152 (1992)).

Pasteurization and other physical-chemical techniques have been used toremove or inactivate blood components, but most of these techniques havebeen limited to fresh plasma or fresh-frozen plasma. To date, thesetechniques are not suitable for treating cellular components of wholeblood.

One disinfectant in use for blood products is beta-propiolactone.Beta-propiolactone, however, is a known carcinogen and hence potentiallyvery dangerous. To the extent that significant residual amounts of thismaterial may remain in the blood product which is actually transfused,the use of propiolactone represents a potential hazard.

There is described in U.S. Pat. No. 4,833,165 (issued on May 23, 1989,in the name of Allan Louderback) the use of as little as 0.1%formaldehyde and/or phenol to inactivate HTLV-III in blood. However,recently available data and information indicate that red blood cellstreated with as little as 0.02% formaldehyde and 0.01% phenol are notviable and not suitable for transfusion.

Viral inactivation by stringent sterilization has not found acceptancesince this method typically destroys erythrocytes, thrombocytes, and thelabile plasma proteins, such as clotting factor VIII. Viable RBC's canbe characterized by one or more of the following: capability ofsynthesizing ATP; cell morphology; P₅₀ values; filterability ordeformability; oxyhemoglobin, methemoglobin and hemochrome values; MCV,MCH, and MCHC values; cell enzyme activity; and in vivo survival. Thus,if virally inactivated cells are damaged to the extent that the cellsare not capable of metabolizing or synthesizing ATP, or the cellcirculation is compromised, then their utility in transfusion medicineis compromised.

Viral inactivation by stringent steam sterilization is not acceptablefor the above reasons. Dry heat sterilization, like wet steam, isharmful to blood cells and blood proteins at the levels needed to reduceviral infectivity. Use of stabilizing agents such as carbohydrates doesnot provide sufficient protection to the delicate blood cells andproteins from the general effects of exposure to high temperature andpressure.

Methods that are currently employed with purified plasma proteinfractions, often followed by lyophilization of the protein preparation,include treatment with organic solvents and heat or extraction withdetergents to disrupt the lipid coat of membrane enveloped viruses.Lyophilization (freeze-drying) alone has not proven sufficient toinactivate viruses, or to render blood proteins sufficiently stable tothe effects of heat sterilization. The organic solvent or detergentmethod employed with purified blood proteins cannot be used with bloodcells as these chemicals destroy the lipid membrane that surrounds thecells.

Another viral inactivation approach for plasma proteins firstdemonstrated in 1958 has involved the use of a chemical compound,beta-propiolactone, with ultraviolet (UV) irradiation. This method hasnot found acceptance in the United States due to concern over thetoxicity of beta-propiolactone in the amounts used to achieve somedemonstrable viral inactivation and also due to unacceptable levels ofdamage to the proteins caused by the chemical agents. Concern has alsobeen raised over the explosive potential for beta-propiolactone as well.Attempts to inactivate viral decontaminants using photosensitizers andlight have also been developed using some non-psoralen photosensitizers.The photosensitizers that have been employed are typically dyes.Examples include dihematoporphyrin ether (DHE), Merocyanine 540 (MC540)and methylene blue.

It would be highly desirable to be provided with anti-pathogeniccompositions for use in blood and blood products, and blood containers,such as a blood donation bag.

It would be highly desirable to be provided with a method for processingand disinfecting human blood products.

It would be highly desirable to be provided with a method fordisinfecting whole blood, blood cells, plasma proteins, and plasma sothat they may be used safely and effectively for diagnostic, therapeuticor research purposes.

It would be highly desirable to be provided with a method vis the bloodcirculatory system for controlling the pathological state produced by apathogen, typically a viral, bacterial, protozoan, fungal or parasiticagent.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide an anti-pathogeniccomposition for use in blood and blood products, and blood containers,such as a blood donation bag.

Another aim of the present invention is to provide a method forprocessing and disinfecting human blood products.

Another aim of the present invention is to provide a method fordisinfecting whole blood, blood cells, plasma proteins, and plasma sothat they may be used safely and effectively for diagnostic, therapeuticor research purposes.

Another aim of the present invention is to provide a method vis theblood circulatory system for controlling the pathological state producedby a pathogen, typically a viral, bacterial, protozoan, fungal orparasitic agent.

In accordance with the present invention, there is provided, ananti-pathogenic composition for use in disinfecting fluids andbiological tissues and surfaces contaminated with fluids and/orbiological tissues, which comprises an anti-pathogenic amount of atleast one quaternary ammonium compound in association with an acceptablecarrier.

A preferred anti-pathogenic composition of the present invention furthercomprises a bisguanidine compound.

In a preferred anti-pathogenic composition of the present invention, thequaternary ammonium compound comprises a mixture of two or morequaternary ammonium compounds.

The fluids and biological tissues may be selected from the groupconsisting of water, water-based solutions, blood (i.e. whole blood) andblood products, skin and organs for transplantation.

The surfaces are selected from the group consisting of containers,filters, tubing, seals, clamps, transfer leg closures, medical devices,operating tables, and medical examination tables.

The containers may be biological fluid containers or water containers.

The container is a blood or blood product container, such as a blooddonation bag or bottle.

The blood product may be selected from the group consisting of plasma,packed red cells, platelet-rich plasma, platelet concentrate andcryoprecipitate.

The composition may be in the form of a cream, ointment, lotion, gel,powder, detergent, soap, liquid, or solid.

In accordance with the present invention, there is provided, a method ofinactivating a pathogen in blood or blood product in a container,comprising contacting at least one of said blood, blood product andcontainer with a composition of the present invention.

In accordance with the present invention, there is provided, a medicaldevice comprising at least a surface treated with an anti-pathogeniccomposition of the present invention or containing at least ananti-pathogenic composition of present invention.

The medical device may be a biological fluid container, such as blooddonation bag or bottle, an in-line filter through which blood or bloodproduct is passed prior to delivery to an individual, and an in-linefilter through which blood or blood product is passed at the point ofcollection from an individual.

A preferred medical device of the present invention, further comprises aporous medium for dispensing said anti-pathogenic composition.

In accordance with the present invention, there is provided a method fortreating in vitro or ex vivo biological fluid, comprising collecting abiological fluid in a container, and exposing the biological fluid to ananti-pathogenic composition of the present invention.

In accordance with the present invention, there is provided a method forinhibiting in vitro or ex vivo infection or replication of humanimmunodeficiency virus in a biological fluid, comprising treating saidbiological fluid with an effective inhibiting amount of a bis-guanidinecompound or a derivative thereof, and at least one quaternary ammoniumcompound in combination with a pharmaceutically carrier, such as DMSO.

In accordance with the present invention, there is provided a method forin vitro or ex vivo disinfecting red blood cells, said processcomprising the steps of:

-   a) contacting said red blood cells with a disinfecting composition    for a period of time sufficient to inactivate pathogen present in    said red blood cells, wherein said disinfecting composition    consisting essentially of a disinfecting concentration of a    bis-guanidine compound in association with a solution of an isotonic    effective concentration of solute, whereby said disinfecting    composition substantially isotonic with blood; and;-   b) isolating said blood cells from said disinfecting composition.

In a preferred method of the present invention, the bis-guanidinecompound is selected from the group consisting of quaternary ammoniumcompounds, and combinations thereof.

A preferred method of the present invention, further comprises removingthe composition after inactivating the pathogen, or removing thecomposition after treating the biological fluid.

This invention relates to a system for processing blood donated for thepurpose of therapeutic transfusion of blood components and,particularly, to improved methods and apparatuses for preparingpathogen-free or pathogen-inactivated blood or a blood component. Thisinvention also relates to a biological fluid processing system forprocessing treated biological fluid into its various components.

The present invention is directed to compositions and methods forreducing the level of pathogens, e.g., infectious viruses and/orbacteria, that may be contained in a red blood cell composition.Specifically, the inventors of the present invention have found thatexposing the red cell composition to a bisguanidine compound in or witha composition having one or more tension-active surfactants, such asquaternary ammonium compounds, inactivates the pathogen(s) sufficientlyto allow the blood or blood product to be transfused, without damagingred blood cells or other blood constituents.

All references cited herein are incorporated by reference in theirentirety.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the effective and safe disinfection ofwhole blood and blood products. The invention has wide application toall blood products such as whole blood for transfusion, blood cells;blood plasma and blood plasma proteins. Since whole blood is rarelyused, the present invention is more particularly directed to processesfor disinfecting compositions containing red blood cells and/or bloodplasma.

The present invention is a composition containing one or moretension-active surfactants as an active agent, such as a quaternaryammonium compound, suitable for use to disinfect blood or a bloodproduct.

The present invention is also a method for treating blood or a bloodproduct involving contacting the blood or blood product with acomposition containing one or more compounds or active agents of thepresent invention, said compound being present in an amount sufficientto inactivate one or more pathogens in the blood, and allowing thecompound to inactivate pathogen(s). The method may further includeremoving the compound from the blood or blood product.

The present invention may also be applied to viral inactivation oftissues and organs used for transplantation, and used in topical creams,ointments, lotions, gels, powders, liquids, solids, detergents or soapsfor treatment of skin or epithelium disorders or for topicaldecontamination.

The present invention may also be used in the manufacture of viralvaccines for human or veterinary use, particularly to produce live,non-viable or attenuated viral vaccines.

The present invention includes a method for reducing viral, bacterial,protozoan, fungal and other parasitic contamination from a biologicalsolution. Biological solutions include, but are not limited to,solutions comprising blood, a blood component, cell culture or acomponent of a cell culture. The method comprises mixing the compositionin a liquid state with a composition of the present invention capable ofbinding to the viral, bacterial protozoan, fungal or parasiticcontamination.

The present invention is also a method for reducing viral, bacterial,protozoan, fungal and other parasitic contamination from medicaldevices, such as those employed by a doctor, nurse, medical techniciansuch as one who collects blood or blood products, or dentist involvingcontacting the medical device with a composition containing one or morecompounds or active agents of the present invention, said compound beingpresent in an amount sufficient to inactivate one or more pathogens inor on the medical device, and allowing the compound to inactivatepathogen(s). The method may further include removing the compound fromthe medical device.

The term “medical device” is intended to mean any tool employed in thechecking, cleaning, collection of fluid from or medical intervention ofan animal or human body. Such tools include, without limitation,surgical instruments such as, but not limited to, probes, scalpels,clamps, forceps, needles, suction devices for removing saliva or bloodincluding all nozzles, seals, tubing, filters, containers and reservoirstherein, endoscopes, optical fibers, transducers, wire, surgical loops,and in-line and out-line tubing and filters through which blood or bloodproduct is passed prior to delivery to an individual and/or at the pointof collection from an individual.

The present invention is also a method for reducing viral, bacterial,protozoan, fungal and other parasitic contamination from water or watercontainers such as found in swimming pools, hot-tubs, Jacuzzis, baths,and whirlpool baths, air-conditioners and humidifiers, involvingcontacting the water or water container with a composition containingone or more compounds or active agents of the present invention, saidcompound being present in an amount sufficient to inactivate one or morepathogens in the water or on the water container, and allowing thecompound to inactivate pathogen(s). The method may further includeremoving the compound from the water or water container. Such water andwater containers can be those found in the home, a hotel, spa or resort,office, or storage building.

The present invention also includes one or more compositions specificfor a particular blood type.

The present invention is also a blood container, or the like, e.g., ablood or blood product transfusion bag, containing one or more compoundsof the present invention.

In the most preferred embodiments of the invention, a compositioncontaining quaternary ammonium compounds as the active agent, alone orin a mixture, are mixed with blood or a blood product, and used todisinfect the blood. The composition is effective for treating blood,blood products, biological tissues, and other biological fluids. Theactive agent is useful against a wide variety of pathogens, includingbut not limited to viruses, bacteria, protozoa, parasites, fungi, andother pathogens. A most preferred composition includes one or morequaternary ammonium compounds, and, optionally, a bis-guanidinecompound.

In accordance with the preferred method for disinfecting blood products,a method is provided in which viruses and bacteria including the HIVviruses, in whole blood and blood products are inactivated. Oncedisinfected the blood and blood products may be used for therapeutic ordiagnostic purposes in a safe and effective manner. The invention isbased upon the unexpected discovery that the compounds do not lyse redblood cells or cause harm to blood products, and are therefore useful asdisinfectants to remove pathogens from the blood or blood product.Moreover, disinfectant compositions containing these compounds which arenot isotonic with respect to blood, and which until now have not beenconsidered for use with blood products, can be used to disinfect plasmaand plasma proteins without denaturing the protein or otherwise causinga substantial loss in physiological activity.

In accordance with the present invention, methods for disinfecting wholeblood or blood products are provided which include the steps ofproviding a disinfectant composition of a disinfecting concentration ofan active agent or compound and a diluent, and then mixing whole bloodor blood product with the disinfecting composition for a length of timesufficient to inactivate any pathogen present in the blood or bloodproduct. The mixing step may be performed by mixing the blood with theconstituents of the composition separately, mixing directly with acomplete composition, or by timing the additional of one or moreconstituents of the composition. After the blood or blood product isdisinfected, the active agent or compound may be optionally separatedfrom the disinfecting composition, providing blood or blood productwhich is safe and effective for therapeutic or diagnostic use.

The term “pharmaceutically acceptable carrier” is intended to mean anycarrier which may be used to solubilize and/or dilute said quaternaryammonium compound in an efficient anti-pathogenic concentration,including without limitation, saline, buffer, DMSO and dextrose.

The term “bis-guanidine compounds” and “quaternary ammonium compounds”is intended to mean all quaternary ammonium compounds and theirderivatives, including without limitation, didecyl dimethylammoniumchloride, all diaminopropyl laurylamine compounds, nonoxynol-9compounds, bis-guanidine compounds and their derivatives. The term“compounds” shall also include any of the chemicals noted above, aloneor associated with another chemical or substance, e.g., in mixingsolutions, including but not limited to DMSO. The preferred activeagents are alkyl trimethyl quaternary ammonium chlorhexidrine (and theirhalogens); dialkyl dimethyl benzyl quaternary ammonium chlorhexidrine.Exemplary quaternary compounds are described in Marchisio, et al., J.Biomater. Sci. Polymer Edn. 6:533-539 (1994), and Cadwallader, et al.,J. Pharm. Sci., 7:1010-1012 (1965), both incorporated by reference intheir entirety.

The composition containing the active agent may also include one or moreadditional compounds, as desired. These compounds may include DMSO, orother agent that changes the polarity of the cell surface; one or moreamino acids; a diluent; one or more chlorides, such as silver chloride;and/or EDTA.

As described below, the above identified compounds will effectivelydisinfect blood and blood products without substantial loss ofphysiological activity. It is also contemplated as being within theteachings of the present invention to disinfect blood or blood productswith oxidizing compounds having sufficient oxidizing properties toinactivate pathogens, such as viruses, bacteria, protozoa, fungi, andparasitic agents.

The term “fluid” is intended to mean water, water-based solutions andbiological fluid.

The term “biological tissue” is intended to mean skin and organs usedfor transplantation.

As used herein, biological fluid refers to any fluid that may betransfused directly into a patient or into the circulatory system of thepatient. Typical biological fluids include but are not limited to: wholeblood; anti-coagulated whole blood (AWB); packed red cells obtained fromAWB; platelet-rich plasma (PRP) obtained from AWB; platelet concentrate(PC) obtained from AWB or PRP; plasma obtained from AWB or PRP; redcells separated from plasma and resuspended in physiological fluid; andplatelets separated from plasma and resuspended in physiological fluid.Blood product or biological fluid also includes any treated or untreatedfluid associated with living organisms, particularly blood, includingwhole blood, warm or cold blood, and stored or fresh blood; treatedblood, such as blood diluted with a physiological solution, includingbut not limited to saline, nutrient, and/or anticoagulant solutions; oneor more blood components, such as platelet concentrate (PC),platelet-rich plasma (PRP, platelet-free plasma, platelet-poor plasma,plasma, or packed red cells (PRC); analogous blood products derived fromblood or a blood component or derived from bone marrow. The biologicalfluid may include leucocytes, or may be treated to remove leucocytes. Asused herein, blood product or biological fluid refers to the componentsdescribed above, and to similar blood products or biological fluidsobtained by other means and with similar properties. In accordance withthe invention, each of these blood products or biological fluids may beprocessed in the manner described herein.

As used herein, pathogen refers to one or more microorganisms or thelike that cause infection. Exemplary pathogens include, but are notlimited to a virus, bacteria, parasite, protozoa or fungus. An exemplaryvirus includes, but is not limited to Herpes simplex virus, HIV,hepatitis, hepatitis A, hepatitis B, hepatitis C, Respiratory syncycialvirus, blue tongue virus, and bovine diarrhea virus. Virus also includesCytomegalovirus, Epstein-Barr virus, Herpes Simplex type I and IIviruses, and other viruses that circulate freely in the blood, as wellas cell-associated viruses. An exemplary bacteria includes, but is notlimited to, Heliobacter pylori, E. coli, Pseudomotias strains, includingaeruginosa, Staplhylococcus, Proteus vulgaris, and Candida albicans. Anexemplary fungus includes, but is not limited to. Aspergillus. Typicalparasites include, but are not limited to: Ameoba, Plasmodiunm,Leishmania, Mycosus profundus, Trypanosoma, Spirochete, and Arbovius.

In a preferred embodiment of the invention, the pathogen being treatedis one that is suited to being inactivated while in blood or a bloodproduct. Reducing the level of infectious pathogen, or similar words,refers to destroying and/or inactivating all or substantially all of theinfectious pathogen. In a preferred embodiment of the invention,reducing the level of infectious pathogen refers to destroying orinactivating the pathogen sufficiently so that the blood or bloodproduct may be transfused or otherwise used.

In accordance with the present invention, suitable diluents include anyof a number of compounds used in the preparation of isotonic solutions.Exemplary solutes include but are not limited to sugars such as dextroseand glucose, polysaccharides such as dextran, albumin, and salts ofalkali earth metals including sodium chloride, potassium chloride, andpotassium bromide. Combinations of solutes known for their utility instoring physiological cells and tissue are also suitable and includesuch combinations as citrate-phosphate-dextrose,citrate-phosphate-dextrose-adenine, andsaline-mannitol-dextrose-adenine. As described in greater detail below,the presence of at least one solute in the diluent in the form of asugar is preferred because sugar contributes to the reduction of anymethemoglobin, oxidized hemoglobulin, formed during the disinfectingprocess.

Diluents having utility in the practice of the present invention fortheir isotonic characteristics can be combined. Combining diluents isparticularly suitable when disinfecting red blood cells becausecommercial collective units of red blood cells are frequently stored inisotonic solutions containing anti-coagulant, such as ACID(acid-citrate-dextrose), CPD (citrate-phosphate-dextrose), CPD-A(CPD-adenine). Thus, when disinfecting collective units of red bloodcells stored in isotonic solutions of anti-coagulant, the disinfectantcomposition may be prepared in a different isotonic diluent, e.g.,normal saline, and combined with the anti-coagulant solution.

Further in accordance the present invention, processes for disinfectingplasma or plasma products, such as plasma protein fractions, optionallyutilize disinfecting compositions having no solute and in which thediluent is sterile water, water, distilled water or sterile anddistilled water. Because plasma and plasma products do not containtissue or other forms of cellular material, there is no compelling needto have an isotonic medium for maintaining cellular osmotic pressure.

Mixing a disinfectant composition with blood or blood products can beperformed by simply combining the blood or blood product anddisinfectant composition in a suitable container with light agitation toassure sufficient interaction between the blood and disinfectantcomposition. Suitable containers include but are not limited to bloodcollection bags and blood storage apparatus. It is preferable, however,to utilize automated cell washing equipment known in the art andavailable from a variety of sources including Cobe.

In accordance with the present invention disinfecting effectiveconcentrations of quaternary ammonium compounds, preferably quaternaryammonium associated with bis-guanidine, and sufficient periods of timefor disinfecting blood and blood products are primarily dependent uponthe choice of compound. It can also be appreciated that usefulconcentrations of each constituent of the composition and periods oftime for disinfecting are interdependent. Thus, compound concentrationscan be varied and a relatively small concentration of one or more activeagents can be a disinfecting effective concentration when mixed withblood or blood products for longer lengths of time. Conversely, whenrelatively larger concentrations of an agent are utilized indisinfectant compositions, the period of time sufficient to disinfectblood or blood products is less.

An exemplary process for disinfecting blood or a blood product mayinclude first mixing the blood with DMSO, agitating, adding an amount ofa surfactant active agent such as quaternary ammonium or bis-guanidinein a dose so that the active agent does not precipitate, and agitating.The process may optionally include a further step of removing the activeagent from the blood composition.

The invention also involves a method for collecting and processing bloodcomprising collecting blood or a blood product in a container;optionally removing or separating a blood product, e.g., plasma, andcontacting the blood or blood product with a composition containing anamount of active agent sufficient to inactivate a pathogen in the bloodor blood product.

The containers which are used in the biological fluid processingassembly may be constructed of any material compatible with a biologicalfluid, such as whole blood or a blood component, and capable ofwithstanding a centrifugation and sterilization environment. A widevariety of these containers is already known in the art. For example,blood collection and satellite bags are typically made from plasticizedpolyvinyl chloride, e.g. PVC plasticized with dioctylphthalate,diethylhexylphthalate, or trioctyltrimellitate. The bags may also beformed from polyolefin, polyurethane, polyester, and polycarbonate.

As used herein, the tubing may be any conduit or means which providesfluid communication between the containers, and is typically made fromthe same flexible material as is used for the containers, preferablyplasticized PVC. The tubing may extend into the interior of thecontainer, and may be used as a siphon, for example. There may be anumber of tubes providing fluid communication to any individualcontainer, and the tubes may be oriented in a number of ways. Forexample, there may be at least two tubes oriented at the top of thecollection bag, or at the bottom of the bag, or a tube at each end ofthe bag.

A seal, valve, clamp, transfer leg closure, or the like is typicallylocated in or on the tubing. It is intended that the present inventionis not limited by the type of material used to construct the containersor the conduit which connects the containers.

A number of additional containers may be in communication with thebiological fluid processing system, and can be utilized to definedifferent flow paths. For example, an additional satellite bagcontaining physiological solution may be placed in communication withthe biological fluid processing system.

In accordance with the invention, the biological fluid collection andprocessing assembly should be able to withstand rigorous sterilizationand centrifugation environments, typically consisting of radiationsterilization (at about 2.5 megarads), and/or autoclaving (at about 110°C. to about 120° C. for about 15 to 60 minutes), and/or centrifugation(typically about 2500 to 3500 G for about 5 to 15 minutes; however,depending on which biological fluid component is intended to havemaximum recovery, the centrifugation may be about 5000 G for about 10 to20 minutes).

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

EXAMPLE 1 Methodology of a Test for Inhibition of HIV-1 Viral Activity

One can screen the bis-guanidine for inhibition of HIV using variousexperimental techniques. One technique involves the inhibition of viralreplication in human peripheral blood mononuclear (PBM) cells. Theamount of virus produced is determined by measuring the quantity ofvirus-coded reverse transcriptase (an enzyme found in retroviruses)which is present in the culture medium. Another technique involvesmeasuring inhibition of purified reverse transcriptase activity in acell free system. Examples of methods for screening which are known tothose skilled in the art are described in more detail as follows.

Methodology for Testing Antiviral Drugs for Inhibition or Replication ofHIV-1 in Human Peripheral Blood Mononuclear (EBM)

Cells from HIV-1 and B virus seronegative donors are isolated byFicoll-Hypaque discontinuous gradient centrifugation at 1,000 times/gfor 30 minutes, washed twice in PBS and pelleted at 300 times/g for 10minutes. Before infection, the cells are stimulated byphytohemagglutinin (PHA) at a concentration of 16.7 μg/ml for three daysin RPMI 1640 medium supplemented with 15% heat-inactivated fetal calfserum, 1.5 mM L-glutamine, penicillin (100 U/ml), streptomycin (100μg/ml), and 4 mM sodium bicarbonate buffer.

HIV-1 (strain LAV-1) is obtained from the Center for Disease Control,Atlanta, and propagated in PHA-stimulated human PBM cells using RPMI1640 medium as above without PHA and supplemented with 7% interleukin-2(Advanced Biotechnologies, Silver Spring, Md.), 7 μg/ml DEAE-dextran(Pharmacia, Uppsala, Sweden), and 370 U/ml anti-human leukocyte (alpha)interferon (ICN, Lisle, III.). Virus is obtained from cell-free culturesupernatant and stored in aliquots at −70° C. until used.

Medium Used for Cellular Culture

The study of cytotoxicity having been conducted on a line T, the culturemedium corresponds to RPMI 1640 supplemented by 10% fetal calf serum(BIO-LAB), 1% glutamine (GIBCO), 1% antibiotics (PSN, GIBCO).

For the study of the residual infectious strength after treatment ofviral preparations, the culture medium of normal human lymphocytes T(complete medium) corresponds to the above medium to which are added 10%interleukin II, polybrene (2 mg/ml) and human anti-interferon serum(1.2500 (M. A. Rey et al), Biochem. Biophys. Res. Corn., 1984, 121126-133).

Cells

The cytotoxicity is studied on a CEM cell clone, line of immature Tlymphocytes.

Infectivity is studied on T lymphocytes obtained from peripheral bloodof a healthy human donor and separated on a Ficoll gradient.

The lymphocytes are stimulated for 3 days by phytohaemaglutinin P (PHAP) diluted to 1/500 and cultivated in a complete medium. The blasticcells T are infected by the virus, treated or not, in order to show upresidual infectious strength.

HIV-1 Virus

The viruses obtained from a supernatant culture of an HIV-1 infected CEMline. This T lymphoblastoid line infected by HIV-1 is used for viralproduction.

The titer of the supernatant substances used is evaluated afterquantitative analysis of the reverse transcriptase activity (ATI) of thevirus.

In the following tests, a supernatant substance was used, the ATI ofwhich was 1.8×10⁶ cpm/ml.

Methods

Study of the Cytotoxicity of Compounds for T Cells

Cells: cellular suspension of the CEM clone with 1×10⁶ cells/ml.

Products: various dilutions in the medium RPMI 1640 are prepared fromthe compounds which was a QACs, Bardac22®.

On a cellular culture plate with 24 wells, 0.5 ml of each of thedilutions are incubated with 0.5 ml of cellular suspension of the CEMclone (5×10⁵ cells) in an incubator at 37° C.

Cellular growth is evaluated after counting the cellular suspensionswith Tryptan Blue® in comparison with a control comprising untreatedcells.

Action of the Bardac, Alone or in Association on the Infectious Capacityof the HIV-1 Virus for Normal Human Lymphocytes T (MLC)

Preparation of the viral solution: several tubes containing 1 ml viralsupernatant, ATI 1.8×10⁶ cmp/ml are, concentrated byultracentrifugation. The resulting virus residues are taken up either byeach of the different dilutions of QACs produced in sterile bidistilledwater or by water (untreated control virus).

Treatment of viral preparations: the concentrated viral preparations areall treated according to the following procedure:

Treated virus samples: the virus residues concentrated byultracentrifugation are resuspended in 100, μl of each of theconcentrations in terms of products (QACs) studied and incubated for 10minutes.

Control viral sample: untreated control virus: the product is replacedby 100 μl of sterile bidistilled water.

Following this 10 minute incubation time, the virus present in eachsample is rinsed and then reconcentrated by addition of 12 ml of RPMI1640 medium and ultracentrifugation. The virus residues are then takenup in 1 ml of complete medium and used for infecting normal human Tlymphocytes.

Measurement of the infectivity of the samples: 4×10⁶ normal human Tlymphocytes are infected with each of the treated viral preparations orcontrols mentioned hereinabove. Infection of the T cells is carried outaccording to a previously described method (F. Barre Sinoussi et al.Science, 1983, 220:868-871): to sum up, 4×10⁶ cells in suspension in 1ml of complete medium are incubated with 1 ml of each of the treatedvial preparations or controls for 1 hour at 37° C.

After two washings using several millilitres of culture medium, thecellular suspensions are adjusted to the concentration of 1×10⁶ cellsper ml. The day of the infection is regarded as day 0.

Control cells: 4×10⁶ T lymphocytes are cultivated and are passed every 3or 4 days under the same conditions as all the other samples.

This control corresponds to the negative control from viral productionduring the course of handling.

Viral production during the course of time is determined by measuringthe reverse transcriptase activity present in the culture supernatantsusing the procedure described below.

Measurement of the Reverse Transcriptase Activity of the Control ViralPreparations and the Viral Samples Treated by the Products

This quantitative analysis makes it possible to evaluate the presence ofnoninactivated residual virus in the staring sample. It is carried outon a basis of 1 ml of culture supernatant taken every 2 or 3 days andultracentrifuged.

The enzymatic activities are measured in 50 μl of a reaction mixturecontaining 50 mM Tris pH 7.8, 20 mM MgCl₂, 20 mM KCl, 2 mMdithiotreitol, 0.25 OD/ml, oligo dT, 0.25 OD/ml poly rA, 0.1% Triton×100and 2.5 μCi 3H DTTP.

After incubation for 1 hour at 37° C., the reaction is stopped by theaddition of 10 μl of 0.2 M EDTA and the samples are deposited on DE81membranes.

After several washings using 5% Na₂HPO₄, then by distilled water, themembranes are dried and the radioactivity measured by means of a pscintillation counter (PACKARD).

Results

Table I shows the effect of a treatment of viral samples by variousproducts on the infectivity of the HIV-1 virus.

TABLE I Quantitative Analysis Of ATI in the Supernatant Substances OnInfected Cultures (cpm/ml) J3 J6 J10 J13 J17 J20 J23 J27 J30 Bardac ®(%) 0.01 334 222 1768 1592 190 390 1328 780 1000 0.006 320 326 204 248364 180 328 588 196 0.004 346 298 160 170 350 218 444 244 264 0.002 4642472 16066 24902 77754 75554 101394 80560 64692 0.0004 702 18136 11732674214 50674 22478 13806 7800 4426 Chlorhexidine (%) 0.01 262 274 200 250230 272 456 420 276 0.005 360 374 580 742 1902 1126 1226 528 720 0.002252 352 1304 1752 4288 6138 4614 1616 4528 Bardac ® + Chlorhexidine (%)0.006 + 0.01 336 202 116 226 280 236 342 510 240 0.006 + 0.005 326 196210 384 274 166 280 406 304 0.006 + 0.002 286 240 196 282 192 274 376478 330 0.004 + 0.01 302 326 210 222 240 350 366 278 248 0.004 + 0.005298 288 206 344 170 346 288 456 282 0.004 + 0.002 228 268 208 232 1200274 254 422 250 0.002 + 0.01 666 262 222 200 244 394 650 322 280 0.002 +0.005 364 246 314 308 4386 308 268 446 254 0.002 + 0.002 998 222 666 908748 1206 1110 370 336 0.0004 + 0.01 2163 234 186 226 204 250 344 352 2260.0004 + 0.005 438 246 824 1142 2340 2190 2120 1094 1466 0.0004 + 0.002576 3362 21734 36246 39364 40126 18480 6530 10476 T1 1516 3120 2482252912 49884 30540 21800 4500 3464 T2 342 4688 61384 64284 69172 1536612364 3500 3790 T3 1594 45068 75046 56928 12872 8932 5468 1050 1746 T4232 220 218 202 190 268 394 244 308 T1: control virus treated by 100 μlof bidistilled water T2: control virus treated by 100 μl bidistilledwater T3: control virus, verification T4: control cells, not infected

Table I clearly shows the synergy of action of the association ofBardac®/chlorohexidine digluconate at low concentration (concentration0.002+0.002) which shows the absence of virus on the thirtieth day;indeed, at the same concentrations, the two products used by themselvesare not virocidal.

EXAMPLE 2 Virocidal Action of Compositions According to the Invention onHBV Viruses

Action on In Vitro Degradation of the Antigen of Hepatitis B Virus(AgHBs) of the Hepatitis B Virus: Virocide+Human Serum Taken from aPatient with Hepatitis B ( 1/1000th)

TABLE II Mixtures tested Bardac ® (%) + CHG* (%) Bardac ® alone CHGalone 0.2 − 0.25 0.2 0.25 0.1 − 0.1 0.1 0.1 0.04 − 0.05 0.04 0.05 0.2 −0.05 0.04 − 0.25 *CHG corresponds to an abbreviation for chlorohexidinedigluconate

TABLE III Results Virocide tested Residual AgHBs (ratio) Bardac ® 0.2%1.35 Bardac ® 0.1% − CHG 0.1% 2.18 Bardac ® 0.2% − CHG 0.05% 3.07Bardac ® 0.2% − CHG 0.25% 3.23

The mixture comprising 0.1% Bardac® and 0.1% CHG displays interestingactivity even though the dose is reduced by 50% compared with 0.2%Bardac® by itself.

General Conclusion on the Various Mixtures Tested

Only the antigen of the residual virus of hepatitis B makes it possibleto discriminate among the Bardac®-CHG mixtures tested.

It appears that 0.02% Bardac® in association with 0.025% of CHG is aseffective as Bardac® at 0.1%. This reduction by a factor of 5 issignificant and important, particularly in the application of acomposition according to the invention in conjunction with anelastomeric material, Bardac® being particularly toxic to the elastomer.

EXAMPLE 3 Virocidal Action of Compositions According to the InventionAgainst Herpes Simplex Virus (HSV)

Tests In Vitro

Tests Conducted

The viral strain: This is a strain of the herpes virus Hominis type 1,strain Bey (HSV1), it is kept alive on human fibroblastic cells or on KBcells and has undergone a number of important changes on one or othertype of cell.

A viral batch is used which is frozen at −80° C. after obtaining acytopathic effect which reached at least 80% of the cells, without priorcentrifugal treatment, which would have removed most of the cellulardebris from the inoculum; indeed, it appears that the infectivity of thevirus disappears very rapidly when it is extra-cellular and inconsequence one is closer to the reality of in vivo infection when oneuses a viral batch in which the virions can still be found in cellulardebris.

This batch was titrated after distribution into aliquot parts of 0.5 ml,maintained at −80° C., on human fibroblastic cells:

-   -   by dilution of 1 in 10 and inoculations of 8 culture tubes by        dilution: 10⁶ ml; and    -   by inoculation of cultures in Petri dishes (35 mm) and the        addition of anti-HSV1 serum into the supernatant in order to        neutralize the varions which might be released into the liquid        medium, so making it possible to count zones: 100 to 150 units        forming zones per dish inoculated with 0.5 ml of a viral        solution diluted to 10⁻⁴.

Cytotoxicity checks are conducted on the products for cellular culturesused in the tests, that is to say the human fibroblastic cells.

The cells are cultivated on a Dulbecco medium enriched with 10% fetalcalf serum; at the moment of their use, the cells are washed and thesame culture medium is used but without calf serum.

For each product tested or each composition, 10 tubes of cellularculture were used and after inoculation of the product, the tubes werekept under observation for 3 days.

Action Observed with the Various Products by Themselves

Each product was, in succession, used pure and at dilutions of 10⁻¹,10⁻², 10⁻³ and added to culture tubes containing 1 ml of nutrient mediumin a volume of 0.1 ml.

Results

-   -   Bardac® 0.04% solution: not toxic at a dilution of 10⁻³    -   chlorohexidine digluconate, 0.05% solution: no toxicity.        Immediate Virocidal Action Observed

Measurement of activity: 0.2 ml viral suspension is mixed with 0.2 ml ofone of the diluted solutions of the test compound. There is then animmediate dilution of 0.2 ml of mixture at the rate of 10⁻⁴ which stopsthe activity of the product. This dilution of mixture is deposited in avolume of 1 ml on a layer of vero cells cultivated in a Petri dish 6 cmin diameter.

After 1 hour of adsorption, the supernatant liquid is drawn off andreplaced by 5 ml of culture medium with no animal serum but to which hasbeen added 1% of a type 1 polyclonal rabbit serum, anti-herpes type.

This does not interfere with multiplication of the intercellular virusbut neutralizes the particles which pass into the liquid phase duringdestruction of the infected cells; at the end of 4 to 5 days, it ispossible with the naked eye to observe zones of cellular lysis whichextend like a spot of oil, each theoretically corresponding to themultiplication of “a zone-forming unit” (UFP) present in the viraldilution which was deposited on the culture. A zone-forming unit may beroughly evaluated as equivalent to an infecting particle or even avirion.

TABLE IV Number of zone-forming units per milliliter of virus diluted to10⁻⁴ (titration carried out on three culture dishes) on day 4 Bardac ®0.02% + Control virus at Bardac ® 0.02% digluconate 0.025% 10⁻⁴, 10⁻⁵,10⁻⁶ 30/125/45 17/26/16  1/223/51 Bardac ® + Control virus at Bardac ®digluconate 0.05% 10⁻⁶ 26/17/20  9/6/8 14/32/30Period of Appearance of Activity

0.2 ml of the non-diluted viral suspension are mixed with 0.2 ml of eachof two following mixtures:

-   -   0.04% Bardac®+0.05% CHG (digluconate)    -   0.02% Bardac®+0.025% CHG

After incubation for 1, 5 and 15 minutes a drop of mixture is inoculatedinto 5 culture tubes and kept under observation for 5 days.

As a control, the diluted virus is sown in the same way into culturemedium instead of the mixtures.

TABLE V Results observed on day 2 on 5 tubes for the two mixturesmentioned Virus + Cytopathic Control mixture effect (on day 2) virus 2/5 1 minute 5/5 0/5  5 minutes 5/5 0/5 15 minutes 5/5Tests in vivo

Tests conducted on Nude mice, either injected intramuscularly with aneedle steeped in a viral suspension or injected after passage of theinfected needle (that is to say steeped in a viral suspension) throughmicrocapsules such as are described hereinafter (Example 8, sample 1)covered with latex, shown at the passage of the needles through themicrocapsules permits of a quite significant and instant level of theinfection of the animals.

The operative procedure is similar to that in Example 8 except for theculture media which must be suitable for the herpes virus.

EXAMPLE 4 Bactericidal Action of Compositions

Test I

Materials

-   -   Micro-organisms tested        -   reference strains            -   Pseudomonas aeruginosa CIP A22            -   Escherichia coli CIP 54127            -   Staphylococcus aureus CIP 5314 Oxford strain        -   hospital strains:            -   Pseudomonas aeruginosa            -   Escherichia coli            -   Staphylococcus aureus            -   Proteus vulgaris            -   Candida albicans    -   Millipore membranes: HAE P047SO (0.45 μm)    -   Mueller Hinton 2 gelose: 4 3301 (Biomerieux)    -   sterile and apyrogenic distilled water (biosedra)        Procedure        Maintenance of the Strains

Each strain of bacteria underwent three sub-culturings on Mueller Hintongeloses at 24-hour intervals prior to use.

Preparation of the Bacterial Inocula

-   -   initial bacterial suspension: A

Prepared on the basis of a bacterial isolation, it must correspond to anopacity equivalent to point 1 on the McFarland scale. It will be usedfor the final tests.

-   -   suspension used for controls and preliminary tests: B

It is prepared by successive dilutions of suspension A down to 10⁻⁶.

Control Group

1 ml of suspension B is sown “en masse” with a Mueller Hinton gelose

Control Filtration Counts

1 ml of suspension B is filtered over a millipore membrane and washedwith 50 ml distilled water.

Enumeration of colonies after 24 hours at 37° C.

Preliminary Tests

For each of the four antiseptic mixtures and for each bacterial strain,the following test was conducted:

-   -   filtration of 1 ml of antiseptic mixture    -   washing (twice in 50 ml of distilled water)    -   filtration of 1 ml of suspension B    -   rinsing using 50 ml of distilled water    -   enumeration of colonies on the membrane after 24 hours at 37° C.        Final Tests

Each antiseptic mixture at double concentration is placed for 1, 5 and10 minutes in contact with 1 ml of suspension A and then filtered.

After the washing stages defined by the preliminary test, the membranesare placed at 37° C. for 25 hours.

Interpretation of the Results

In the procedure adopted a mixture intended to be antiseptic is regardedas bactericidal if, after a specific contact time, it reduces thebacterial inoculum by at least 5 logarithms.

This effect is visualized by counting, on the final test membranes, anumber of colonies compared with that observed in the preliminary testscorresponding to the same strain and the same mixture.

Results

TABLE VI Efficacy of Composition at t = 1 min Mixture No. 2 Mixture No.1 Bardac ® 0.04 g/100 ml Strain Bardac ® 4% Digluconate 0.05 g/100 ml S.aureus CIP 53154 E E E. coli CIP 54127 E E P. aeruginosa CPI A22 E E S.aureus E E E. coli E E P. aeruginosa NE E P. vulgaris NE NE C. albicansE E E or NE (Effective or Not Effective)

TABLE VII Efficacy of Composition at t = 5 min Mixture No. 2 Mixture No.1 Bardac ® 0.04 g/100 ml Strain Bardac ® 4% Digluconate 0.05 g/100 ml S.aureus CIP 53154 E E E. coli CIP 54127 E E P. aeruginosa CPI A22 E E S.aureus E E E. coli E E P. aeruginosa NE E P. vulgaris NE NE C. albicansE E E or NE (Effective or Not Effective)

TABLE VIII Efficacy of Composition at t = 10 min Mixture No. 2 MixtureNo. 1 Bardac ® 0.04 g/100 ml Strain Bardac ® 4% Digluconate 0.05 g/100ml S. aureus CIP 53154 E E E. coli CIP 54127 E E P. aeruginosa CPI A22 EE S. aureus E E E. coli E E P. aeruginosa E E P. vulgaris E E C.albicans E E E or NE (Effective or Not Effective)Conclusion

After 1 minute of contact, no mixture was effective on all the strainsstudied. Mixture No. 2 is however the most efficient because only onestrain of Proteus vulgaris appears to be insensitive.

After 5 minutes of contact, mixture No. 2 could be considered aseffective were it not for the same strain of Proteus vulgaris whichresists the product even after 5 mins.

After 10 minutes of contact, mixtures Nos. 1 and 2 give satisfactoryresults over all the strains studied.

Test 2

Another test was carried out a basis of the following solutions

-   -   100 ml of a 0.1% solution of chlorohexidine digluconate (pH 6);        and    -   100 ml of a 0.1% solution of Bardac22®.

The activity of these solutions, alone or in combination, was moreparticularly tested in respect of Staphylococcus aureus ATCC 6538,Escherichia coli ATCC 11229 and Candida albicans ATCC 10231.

The tests were conducted in accordance with the procedure described inChapter I/2.1 and 2.2 of Tichtlinien für die Prüfung und Bewertungchemischer Desinfektionsverfahren der DGHM/Guidelines for Testing andEvaluating Chemical Disinfection Processes of DGHM as at 1.1, 1981.

Tables IX, X and XI respectively illustrate the bactericidal andfungicidal activity of a 0.1% aqueous solution of chlorohexidinedigluconate, a 0.1% aqueous solution of Bardac22® and a mixture of equalparts of both compounds. The procedures of which these Tables illustratethe results comprise incubation of 72 hours at 37° C. and the use of thefollowing inactivating substances as controls:

-   A: 3% Tween®80+0.3% lecithin+0.1% cysteine-   B: 3% Tween®80+3% saponin+0.1% cysteine+0.1% histidine;-   C: 3% Tween®80+0.3% lecithin+0.1% histidine+0.5% sodium    thiosulphate.

In these Tables, the sign “+” indicates an increase and the sign “−”denotes the absence of growth.

The following Table IX which illustrates the bactericidal and fungicidalactivity of a 0.1% aqueous solution of chlorohexidine digluconate (1% ofthe test solution corresponds to 0.001/digluconate), shows theinhibition of the growth of S. aureus and of E. coli by a 0.5% dilutionof the chlorohexidine digluconate solution referred to, corresponding toa concentration of active substance of 0.0005%; C. albicans is inhibitedby a 1% dilution of the said solution (0.001% chlorohexadinedigluconate). In Table X, the didecyl dimethyl ammonium chloride showsan even more pronounced effect of inhibiting gram positive organisms Saureus and C. albicans with a 0.1% dilution-(0.0001% didecyl dimethylammonium chloride) and an inhibition of E. coli (a gram negativeorganism) with a 0.5% dilution (=0.0005% didecyl dimethyl ammoniumchloride) Table X.

Table XI shows that the association of these two antiseptics makes itpossible to inhibit the growth of the aforesaid micro-organisms atsignificantly greater dilutions (action in respect of E coli and C.albicans at concentrations of 0.00025% chlorohexidine digluconate and0.00025% didecyl dimethyl ammonium chloride; action in respect of S.aureus for a concentration of 0.00005% for each of the two aforesaidantiseptics).

TABLE IX Chlorohexidine Digluconate Alone (1% solution → 0.001%digluconate) Test Strain S. aureus E. coli Candida albicans Conc. % ATCC6538 ATCC 11229 ATCC 1031 (vol/vol.) not A B C not A B C not A B C 1.0− + + + − + + + − + + + 0.5 − + + + − + + + + + + +0.1 + + + + + + + + + + + + 0.05 + + + + + + + + + + + +0.025 + + + + + + + + + + + + 0.0125 + + + + + + + + + + + +0.00625 + + + + + + + + + + + +

TABLE X Bardac22 ® Alone (1% solution → 0.001% Bardac22 ®) Test StrainS. aureus E. coli Candida albicans Conc. % ATCC 6538 ATCC 11229 ATCC1031 (vol/vol.) not A B C not A B C not A B C 1.0 − + + + − + + +− + + + 0.5 − + + + − + + + − + + + 0.1 − + + + + + + + − + + +0.05 + + + + + + + + + + + + 0.025 + + + + + + + + + + + +0.0125 + + + + + + + + + + + + 0.00625 + + + + + + + + + + + +

TABLE XI Composition according to the invention (2% solution → 0.001%Bardac ® and 0.001% CHG) Test Strain S. aureus E. coli Candida albicansConc. % ATCC 6538 ATCC 11229 ATCC 1031 (vol/vol.) not A B C not A B Cnot A B C 2.0 − + + + − + + + − + + + 1.0 − + + + − + + + − + + + 0.5− + + + − + + + − + + + 0.1 − + + + + + + + + + + +0.05 + + + + + + + + + + + + 0.025 + + + + + + + + + + + +0.0125 + + + + + + + + + + + + 0.00625 + + + + + + + + + + + +

The tests, the results of which are illustrated in Tables XII, XIII andXIV, were conducted at reaction temperature of 22° C. with an incubationof the sub-cultures 72 hours at 37° C. with, as inactivating substances:3% Tween® 80+3% saponin+0.1% cysteine+0.1% histidine; these Tablesrespectively illustrate the bactericidal and fungicidal activity of a0.1% aqueous solution of chlorohexidine digluconate, of a 0.1% aqueoussolution of Bardac® and a mixture of equal parts of the two compoundsand show the action of the aforesaid antiseptics as a function of thecontact time and cause the appearance of the same synergy of action ofthe association as above, in so far as the association of the twocompounds makes it possible significantly to reduce their concentrationin order to obtain the same effect.

TABLE XII (CHG by itself) Test Strain S. aureus ATCC 6538 E. coli ATCC11229 C. albicans ATCC 1031 organisms/ml organisms/ml organisms/ml 8 ×10⁸ 2 × 10⁹ 8 × 10⁷ Conc. % Disinfection time (minutes) (vol/vol.) 1 3 515 30 60 1 3 5 15 30 60 1 3 5 15 30 60 25 + + + + − − + + + − − − + + +− − − 10 + + + + + − + + + − − − + + + + − − 5 + + + + + + + + + + −− + + + + − − 1 + + + + + + + + + + + + + + + + + −0.5 + + + + + + + + + + + + + + + + + +0.1 + + + + + + + + + + + + + + + + + +0.05 + + + + + + + + + + + + + + + + + +

TABLE XIII (Bardac ® by itself) Test Strain S. aureus ATCC 6538 E. coliATCC 11229 C. albicans ATCC 1031 organisms/ml organisms/ml organisms/ml8 × 10⁸ 2 × 10⁹ 8 × 10⁷ Conc. % Disinfection time (minutes) (vol/vol.) 13 5 15 30 60 1 3 5 15 30 60 1 3 5 15 30 60 25 + − − − − − − − − − − − −− − − − − 10 + − − − − − + + − − − − − − − − − − 5 + + + − − − + + + − −− + + − − − − 1 + + + + − − + + + + + + + + + + − −0.5 + + + + + + + + + + + + + + + + + +0.1 + + + + + + + + + + + + + + + + + +0.05 + + + + + + + + + + + + + + + + + +

TABLE XIV (Composition according to the Invention) Test Strain S. aureusATCC 6538 E. coli ATCC 11229 C. albicans ATCC 1031 organisms/mlorganisms/ml organisms/ml 8 × 10⁸ 2 × 10⁹ 8 × 10⁷ Conc. % Disinfectiontime (minutes) (vol/vol.) 1 3 5 15 30 60 1 3 5 15 30 60 1 3 5 15 30 6025 + − − − − − − − − − − − + − − − − − 10 + + + − − − − − − − − − + − −− − − 5 + + + + − − + − − − − − + + + − − − 1 + + + + + + + + + + −− + + + + + + 0.5 + + + + + + + + + + + + + + + + + +0.1 + + + + + + + + + + + + + + + + + +0.05 + + + + + + + + + + + + + + + + + +

EXAMPLE 5 Spermicidal Action

Procedure

The various samples of sperm (one drop of sperm per sample) are obtainedfrom healthy volunteers.

Measurement of Vitality

One drop of sperm is treated by eosine-nigrosine. A smear is thenprepared and 100 spermatozoids examined with a microscope. The deadcells are coloured red, either totally or partially. The living cellsare colourless.

Substances Studied

The substances studied were Bardac® and chlorohexidine digluconate,tested alone and in association in accordance with the invention. Thevarious stages of the procedure are set out in Tables XV to XVII.

The maximum contact time of the various substances and of the sperm wasconfined to 3 minutes in order to guarantee the effectiveness of themethod under the conditions of use. The concentration of the activesolutions in the sperm was limited to 10% in order to reduce the risksof toxicity of the products in respect of the mucosa.

TABLE XV Bardac Contact time of substances and sperm Concentrations of 1min 3 min solutions in terms Concentrations of the solutions in thesperm Control at TO active substances 5% 10% 5% 10% 82.8 ± 7.1 0.4% 10.7± 7.6    5 ± 6.4 0.8 ± 1.2 0.8 ± 1   81.4 ± 5.9 0.6% 10.5 ± 9.7   9.2 ±12.9 2.2 ± 4.5 0.4 ± 0.9 81.4 ± 5.9 0.8%  9.7 ± 10.2 1.8 ± 2.5 0 0 79.7± 5.3   1% 0 0 0 0

TABLE XVI Chlorohexidine Digluconate Concen- trations of solutions inContact time of substances and sperm Control terms 1 min 3 min at activeConcentrations of the solutions in the sperm TO substances 5% 10% 5% 10%98 ± 6.2% 0.5% 77 ± 10.8 77.2 ± 5.6 78 ± 5.7 75.5 ± 4.8

TABLE XVII Bardac + Chlorohexidine digluconate Contact time ofsubstances and sperm Concentrations of 1 min 3 min solutions in termsConcentrations of the solutions in the sperm Control at TO activesubstances 5% 10% 5% 10% 88.4 ± 5.4% B 0.4% + DC 0.5% 15.6 ± 13.2 5.1 ±8    8.6 ± 10.8 2.4 ± 4.1 B 0.4% + DC 0.8% 21.3 ± 14   6.7 ± 3   18.3 ±13.3 6.3 ± 7.8 B 0.4% + DC 1%   19 ± 11.1  10 ± 8.7  16 ± 9.8  9.3 ±11.9 B 0.6% + DC 0.5% 6.3 ± 11    4 ± 6.9 1.3 ± 2.3   1 ± 1.7 B 0.8% +DC 0.5% 3.7 ± 6.3 0   2 ± 3.5 0

EXAMPLE 6 Study of the Mixture of Bardac22® and ChlorohexidineDigluconate in Aqueous Solution

All the mixtures were prepared in weight/weight percentages in plasticphials kept away from the light and at room temperature (20° C.±1° C.).

Daily observations of these phials made it possible or not to detect thepresence of a white precipitate. This (when it forms) is found on theresidue of the majority phial but also on the walls wetted by theliquid.

Study of 50/50 Solutions of Bardac22® Chlorohexidine at VariousConcentrations

The study was conducted in a first stage on aqueous solutions atdifferent concentrations of active principles but for a mixture of thetwo active principles (Bardac® and chlorohexidine digluconate) in equalproportions (50/50):

-   -   30% solution of active principles—precipitated at 24 hours    -   20% solution of active principles—precipitated at 24 hours    -   10% solution of active principles—precipitated at 24 hours    -   5% solution of active principles—precipitated at 24 hours    -   1% solution of active principles—precipitated at 24 hours        Study of 10 and 20% Solutions of Bardac® and Chlorohexidine in        Different Proportions

In a second stage, the concentrations of active principles were fixed at10% and 20% but the proportions of the two active principles werevaried.

TABLE XVIII Recapitulative Table of the 10% Solutions Of ActivePrinciples Ratio of chlorohexidine chlorohexidine digluconate/digluconate Bardac ® Bardac ® (%) (%) Remarks  0.5/99.5 0.05 9.95 noprecipitation  1/99 0.1 9.9 no precipitation  2/98 0.2 9.8 noprecipitation  4/96 0.4 9.6 no precipitation  8/92 0.8 9.2 noprecipitation 16/84 1.6 8.4 no precipitation 25/75 2.5 7.5 precipitationat 2 months 35/65 3.5 6.5 precipitation at 9 days 40/60 4.0 6.4precipitation at 6 days 50/50 5.0 5.0 precipitation at 24 hours

TABLE XIX Recapitulative Table of the 20% Solutions of Active PrinciplesRatio of chlorohexidine chlorohexidine digluconate/ digluconate Bardac ®Bardac ® (%) (%) Remarks  0.5/99.5 0.1 19.9 no precipitation  1/99 0.219.8 no precipitation  2/98 0.4 19.6 no precipitation  4/96 0.8 19.2 noprecipitation  8/92 1.6 18.4 no precipitation 3.2 16.8 8.4 noprecipitation 25/75 5.0 15.0 precipitation at 2 months 35/65 7.0 13.0precipitation at 9 days 40/60 8.0 12.0 precipitation at 6 days 50/5010.0 10.0 precipitation at 24 hours

EXAMPLE 7 Study of the Mixture of the Bardac 22® ChlorohexidineDigluconate in Alcohol Solution

The dissolutions in the proportions indicated for water are identicalwhen the two products are in solution in glycerol.

EXAMPLE 8 Study of the Disinfectant Capacity in Respect of Bacteria andFungi of Microcapsules Containing a Composition According to theInvention and Included in a Polyvinyl Cloth After the said Tissue hasbeen Punctured by a Needle.

Material and Method

Two samples of microcapsules included in polyvinyl or latex clothscomprise:

-   -   sample 1: microcapsules containing a composition according to        the invention, included in PVDC. The thickness of the cloth        covering the microcapsules is around 500 μm and the total        thickness of the cloth is around 1300 μm and the quantity of        disinfectant available after the cloth has been torn by a needle        (0.5×16 mm) is approx. 1.5 μg;    -   sample 2: microcapsules containing a composition according to        the invention of the even type I, included in the EVA. The        thickness of the cloth covering the micro-capsules is about 500        μm and the quantity of disinfectant available after the cloth        has been torn by a needle (0.5×16 mm) is approx. 0.4 μg.

The microbiological samples for carrying out the test are prepared usingthe following micro-organisms: Escherichia coli ATCC 11229,Staphylococcus aureus ATCC 6538 and Candida albicans ATCC 10231.

The strains are cultivated in a CSL medium (Merck) for 24 hours at 37°C., then dilutions comprised between 10⁻³ and 10⁻⁶ are prepared in a0.9% sodium chloride solution.

One millilitre of each of the various cultures obtained (diluted and notdiluted) is added to 9 ml of a pool of inactivated human serum. Thenumber of micro-organisms per ml of the test solution is mentioned inthe following Table XX.

Three types of needle were used: 0.7×30 mm (22 G×¼″, Terumo), 0.5×16 mm(25 G×⅝″, Terumo), 0.3×13 nun (30 G×½″, Microlance, Becton Dickinson).

These needles are mounted on 1 ml syringes, steeped in a microbialsolution as described above (at least 1 cm of the needle must be steepedin the solution) and 0.1 ml of solution is aspirated. It then piercesthe above-described cloths, placed in Petri dishes covered with casoagar(Merck, medium supplemented by 3% of Tween® 80 and 0.3% lecithin) withthe said needles.

The sites where the agar is inoculated by the needle are marked, thenthe culture media are incubated for 72 hours at 37° C. prior to readingof the results.

Results

Table XX shows the number of positive cultures obtained afterperforation (5 perforations per test) with a 0.3×13 mm (30 G×½″,Microlance, Becton-Dickinson) needle, and culture of the perforatedcloth on a casoagar medium (Merck) supplemented as stipulated above.

TABLE XX Number of positive cultures obtained after perforationMicrobial Sample 1 Sample 2 solution Micro-capsules ControlMicro-capsules Control E. coli 3 × 10⁸/ml + − − − − + + + + − + + + −− + + + + + 2 × 10²/ml − − − − − − − − − − − − − − − − − − − − 10²/ml −− − − − − − − − − − − − − − − − − − − S. aureus 2 × 10⁸/ml + + + −− + + + + + + + + + + + + + + + 2 × 10⁵/ml − − − − − − − − − − − − − − −− − − − − 10²/ml − − − − − − − − − − − − − − − − − − − − C. albicans 3 ×10⁷/ml − − − − − + − − − − + + − − − + + − − − 3 × 10⁵/ml − − − − − − −− − − − − − − − − − − − − 10²/ml − − − − − − − − − − − − − − − − − − −− + growth of micro-organisms − absence of growth

These results show in particular that small concentrations of bacteriaor fungi (10²/ml) are absorbed or eliminated mechanically by thepolyvinyl cloths; a concentration of 105 organisms per millilitre may beregarded as the limit which produces no growth when the smallest needlesare used (9.3×13 mm, Table X; on the other hand, results of the sameorder are obtained with the needles measuring 0.7×30 mm and 0.5×16 mmrespectively. Growth is only obtained for high concentrations ofbacteria (10⁸/ml) or fungi (10⁷/ml), upon perforation of the controlcloths only (sample 1).

These results likewise show that the microcapsules of sample 1 (PVDC)are more effective than the microcapsules of sample 2 (EVA).

Conclusion

The tests conducted with sample 1 show these virtually instantaneousdisinfectant effects of the compositions according to the inventionincorporated into microcapsules under conditions approximating thoseencountered at a hospital.

EXAMPLE 9 Effects of Compounds on the Hemolysis of Erythrocytes

The effect of the compounds on the in-vitro hemolysis of erythrocytes inheparinized whole blood is monitored by a spectrophotometric method.

Various formulations and concentrations of the test compound are addedto heparinized bovine blood and mixed gently in a 1.5 ml microcentrifugetube. The sample is allowed to incubate for 1 hour at 37° C. At the endof incubation, the samples are removed from the incubator andcentrifuged at 2000 rpm for 5 minutes. A 200 μl aliquot of thesupernatant is removed from each sample into a 96 well microtiter plateand read at 413 nm. The absorbance of the test sample is compared to thecontrol sample containing phosphate buffered saline, pH 7.2. Absorbancereading of the test samples that are higher than the control sample aredeemed to be hemolytic.

Table XXI summarizes the concentration and the components of the testcompounds that are non-hemolytic under the test conditions.

TABLE XXI Non-hemolytic Test Compounds Concentration at which hemolysisTest Compound Components is not observed Bardac 22 ® 0.0125%Chlorohexidine Gluconate 0.0125% Bardac 22 ® 0.0125% ChlorohexidineGluconate 0.0125% Decamethonium Iodide 0.0125% Bardac 22 ®  0.001%Chlorohexidine Gluconate  0.001% Tergitol NP-9  0.001% Bardac 22 ®0.0125% Chlorohexidine Gluconate 0.0125% Hexamethonium Bromide 0.0125%Bardac 22 ® 0.0005% Chlorohexidine Gluconate 0.0005% Tri-n-ButylPhosphate 0.0005%

Although the present invention has been described in terms of aparticular preferred embodiments, it is not limited to thoseembodiments. Alternative embodiments, examples, and modifications whichwould still be encompassed by the invention may be made by those skilledin the art, particularly in light of the foregoing teachings.

1. A non-hemolytic method for inhibiting in vitro or ex vivo a pathogenin a blood sample, comprising treating said blood sample with aneffective inhibiting amount of a composition which consists of didecyldimethyl ammonium chloride digluconate and chlorohexidine digluconate,each of which is present at a concentration of 0.002%, in combinationwith a pharmaceutically acceptable carrier, wherein red blood cellsremain intact in said blood sample.
 2. A non-hemolytic method for invitro or ex vivo disinfecting a red blood cell sample, said methodcomprising the steps of: a. contacting said red blood cell sample with adisinfecting composition for a period of time sufficient to inactivatepathogen present in said red blood cell sample, wherein saiddisinfecting composition consists of a composition which comprisesdidecyl dimethyl ammonium chloride and chlorohexidine digluconate, eachof which is present at a concentration of 0.002%, in association with asolution of an isotonic effective concentration of solute, whereby saiddisinfecting composition is substantially isotonic with red blood cells;and b. isolating said red blood cells from said disinfectingcomposition, wherein the red blood cells remain intact.
 3. The method ofclaim 2, further comprising removing the composition after inactivatingthe pathogen.